The objective of this project is to develop and evaluate novel anti-HIV gene therapy strategies for AIDS in a clinically relevant macaque SHIV (simian human immunodeficiency viruses) model. Highly active antiretroviral therapy (HAART) has reduced the morbidity and mortality associated with HIV infection, but the emergence of resistant viral variants, toxicity, and compliance issues are significant obstacles in controlling AIDS. Additionally, recent promising vaccine trials have failed to protect from HIV, emphasizing the importance of developing alternative therapies. One such alternative therapy is the genetic modification of hematopoietic cells to make them resistant to HIV infection or to at least inhibit HIV replication. Here we will explore two main strategies to accomplish the genetic modification. We will evaluate novel lenti and foamy virus vectors and we will study targeted gene insertion to interfere with CCR5 expression. Another key aspect of our proposal is the use of a clinically relevant nonhuman primate AIDS model. We have recently shown that both vector systems allow for efficient gene transfer to HSCs in nonhuman primates, and we have preliminary data of zinc finger mediated CCR5 disruption in macaque cells. Based on these findings we propose 4 aims. In aim 1 we will study combinatorial transgene cassettes that inhibit HIV replication. We will study these cassettes in lentivirus and foamy virus vectors to determine the safety of these vectors systems first in NOD/SCID mice and then in the nonhuman primate setting. These studies will also allow us to determine whether foamy virus vectors can overcome the adverse effect on vector titers with lentivirus vectors for some transgene cassettes. The anti-HIV vectors we propose to test contain MGMT transgenes to allow in vivo selection post-transplantation. These studies will allow us to compare gene marking, engraftment and in vivo selection with these two promising vector systems. In aim 2 we will establish conditions for efficient in vivo selection of gene-modified cells and explore means to reduce the potential for enhancer activation by integrated vector proviruses. In aim 3 we will challenge monkeys that have high marking levels to determine if our gene therapy approach can protect from SHIV infection in vivo. In aim 4 we will develop methods to introduce anti-HIV genes at a defined chromosomal locus using targeted gene insertion in order to avoid the potential for vector-mediated leukemogenesis. This approach has the added advantage that knockout of CCR5 and insertion of an anti-HIV transgene should both increase the resistance of gene-modified cells to SHIV infection. SHIVs contain several HIV genes including tat, rev and env that we will target to inhibit viral replication in our macaque-SHIV model. The proposed studies will allow us to test the gene therapy approach in a highly relevant monkey model and to develop methods for efficient and safe delivery of therapeutic transgenes to HSCs and to T cells. Since the vectors we have developed contain anti-HIV transgenes that function both in the SHIV monkey model and for HIV infection in humans, the proposed studies should thus also provide critical efficacy and safety data for promising vectors for future clinical trials. PUBLIC HEALTH RELEVANCE: The proposed research will evaluate the efficacy and safety of novel gene therapy strategies for the treatment of AIDS using a highly clinically relevant monkey model. If successful the proposed studies will lead to novel approaches to protect individuals from AIDS using stem cell gene therapy.